Angiogenesis is required for embryogenesis, tumor formation, and wound healing. Signaling by binding of vascular endothelial growth factor (VEGF) to its receptor VEGFR-2 is a major control point for angiogenesis. The magnitude of this signaling pathway is intimately associated with the abundance of VEGFR-2 at the cell surface. Although VEGF-induced endocytosis and degradation control the cell-surface level of VEGFR-2, very little is known about the machinery that governs VEGFR-2 endocytosis and degradation. Epsin is an endocytic adaptor protein that mediates endocytosis of activated receptors. We have generated mutant mice that lack epsin 1 and 2 globally or selectively in endothelial cells. These mutant mice exhibited similar embryonic lethality due to abnormal vascular development typified by increased vessel density and disorganized architecture. VEGFR-2 expression was elevated, reflecting enhanced VEGF function. We hypothesize that epsin-dependent regulation of VEGFR-2 endocytosis and degradation is crucial for VEGF-mediated vascular development and angiogenesis. To test this hypothesis, we will utilize endothelial cell-specific epsin double knockout mice to determine the functions of epsins in regulating angiogenic sprouting, endothelial cell proliferation, and endothelial cell survival. We will engineer tamoxifen inducible endothelial cell-specific epsin double knockout mice to probe the role of epsins in postnatal angiogenesis under physiological and pathological conditions. The in vivo studies will be complemented by in vitro assays of endothelial cell migration, proliferation, tube formation, vessel sprouting, and VEGF-dependent signaling. We will also use biochemical and fluorescent imaging techniques to investigate the interaction of epsins and VEGFR-2 and the role of epsins in VEGF-induced VEGFR-2 endocytosis and degradation. The information gained will help us determine molecular mechanisms by which epsin-mediated endocytosis regulates VEGF signaling and controls vascular development and angiogenesis. These studies should contribute to the application of endocytic components to new therapeutic strategies for cardiovascular diseases and cancer.